Human ether go-go related gene (hERG, KCNH2) potassium channels are of extraordinary clinical importance because they play a prominent role in heart where they generate a current that repolarizes cardiac action potentials. Mutations in hERG channels and inhibition by drugs cause a reduction in hERG and account for inherited and acquired forms of a type of heart disease known as long QT syndrome (LQTS) which emphasizes the importance of these channels in normal physiological function. The acquired form of LQT is due to the off-site effects of prescription drugs which inhibit hERG, and are a prevalent and serious clinical problem. hERG channels have highly specialized gating (opening and closing) properties that optimize them for their cellular roles in the heart and specialized subunit assembly properties that also control channel gating. The association of hERG with other regulatory or accessory proteins is also a major area of interest for understanding how hERG channels are regulated. The goal of the proposed experiments is to understand the molecular mechanisms that underlie these specializations and how they control hERG current. We will be testing recent structures showing direct N- and C-terminal domain interactions of hERG that we first showed using biochemical, electrophysiological and fluorescence measurements, and how these domain interactions control gating of the channels. Our approach is cutting-edge as we will use electrophysiological recordings to investigate channel conformational changes and fluorescence microscopy to study how structural interactions control channel gating and regulation. We will take advantage of non-canonical amino acid biology to introduce small probes to hERG and introduce metal binding sites at locations guided by structures and probe for movements with transition metal FRET and voltage. Completion of these studies will lead to a greater understanding of the basic mechanisms for hERG channel gating and insight into how intracellular domains of the channel regulate opening and closing. Our outcomes are anticipated to lead to rational biomedical strategies and new molecular target for the treatment of cardiac arrhythmias.